Ab81887

The expression of PACAP, PAC1, VPAC1, VPAC2, and CGRP in the trigeminal ganglion (TG) and the TNC were measured using an immunohistochemistry assay. To avoid the measuring acute changes, the rats were sacrificed and sampled 24 h after the final inflammatory soup stimulation or normal saline infusion. TNC and bilateral TG were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA). The samples were placed in liquid nitrogen and frozen for 10–15 sec and then were cut into 20 µm-thick serial sections on a freezing microtome (CM1850; Leica, Wetzlar, Germany). The sections were blocked with fresh goat serum, which was mixed with 100 µL goat serum (ZLI-9021, ZSGB-Bio, China), 50 µL 10% TritonX-100, and 850 µL PBS, and incubated in a 37°C incubator for 20 min. Then, the sections were incubated with respective diluted primary antibodies for anti-PACAP (1:80, sc-25439, Santa Cruz Biotechnology, USA), anti-PAC1 (1:400, ab54980, Abcam, USA), anti-VPAC1 (1:50, sc-30019, Santa Cruz Biotechnology, USA), anti-VPAC2 (1:50, sc-30020, Santa Cruz Biotechnology, USA), and anti-CGRP (1:50, ab81887, Abcam, USA) at 4°C overnight. After washing off the primary antibodies, the sections were stained with anti-mouse/rabbit secondary antibodies (1:1, KIT-5030, Maixin Biological Technology, China) at room temperature for 2 h.

The frozen sections were blocked using goat serum and then incubated at 4°C overnight with respective diluted primary antibodies for anti-PACAP (1:50, ab174982, Abcam, USA), anti-PAC1 (1:400, ab54980, Abcam, USA), anti-CGRP (1:50, ab81887, Abcam, USA) and anti-NeuN (1:3000, MAB337, Merck Millipore, Germany). After washing off the primary antibodies, the sections were incubated with goat anti-rabbit (1:500, A-11034, Thermo Fisher, USA) and goat anti-mouse (1:1000, A-21424, Thermo Fisher, USA) at darkroom temperature for 3 h. Immunohistochemistry and immunofluorescence were observed through a 20x magnification of TG and 40x magnification of TNC on a microscope (DP73, Olympus, Tokyo, Japan). Six sections were selected from each rat TG at 20 magnification, and one image was selected from each section. Three sections were selected from each rat TNC at 40 magnification, and bilateral images were selected from each one. The average integrated density of six images was defined as the final result for each animal.

Cells on iBidi treat 8-chamber slides were fixed for 15 min using 4% paraformaldehyde (PFA), washed one time with PBS and permeabilized 10 min using PBS/0.1% Triton X-100. Cells were blocked using PBST (PBS+ 0.1% Tween 20) with 2% Bovine Serum Albumin (BSA). Cells were incubated with primary antibodies (α-dsRNA (SCICONS 10010500), α-TUBB3 (BioLegend 802001), α-Nucleocapsid (GeneTex GTX135357), α-Trk.A (abcam ab216626), α-Trk.B (Rnd Systems AF1494), α-Trk.C (Rnd Systems MAB373), α-CGRP (abcam ab81887), and α-Ret (abcam ab134100)) diluted (1:400) in PBST with 1% BSA overnight in a humidified chamber at 4°C. After 3 washes of 10 min in PBST, cells were incubated with secondary antibody (AlexaFluor – Life Technologies) diluted (1:1,000) in PBST with 1% BSA for 1 hour at room temperature. Stained cells were washed 3 times with PBST containing DAPI and mounted using iBidi mounting medium. Slides were imaged using RPI spinning disk confocal microscope.